We have modified the original protocol developed by Kroll and Amaya (Development 122, 3173-3183, 1996) to improve efficiency.Consult our “previous version” of the transgenic protocol for general background and details of the method, published in Early Development of Xenopus laevis: A Laboratory Manual (Sive, H. L., Grainger, R. M. and Harland, R. M., Cold Spring Harbor Laboratory Press, 2000).The protocol described here is the latest version of our protocol that includes both the modifications originally made for transgenesis in X. tropicalis (Offield, M. F. et al., Development 127, 1789-1797, 2000, Hirsh, N. et al., Dev Dyn. 225, 422-433, 2002) and some further refinements.The efficiency of transgenesis with this protocol is around 10%: ~30% of injected eggs cleave normally, ~70% of normal cleavers gastrulate normally, and ~50% of the remaining normally gastrulated embryos express a transgene.The major changes from previous iterations of the protocol are as follows:

1) Use of oocyte extract instead of egg extract.This modification was originally made for the transgenic procedure in X. tropicalis (Hirsh, N. et al. Dev Dyn. 225, 422-433, 2002), but also works well in X. laevis.

3) The restriction enzyme and MgCl2 may be omitted from the transgenic reaction mix.While the use of restriction enzyme stimulates transgene integration, it can also damage the host genome.Embryos carrying the damaged genome often develop abnormally and will show ectopic transgene expression.If the transgene is shorter than 5 kb, the restriction enzyme and MgCl2 can be omitted from the reaction.However, if the transgene is longer than 10 kb, the restriction enzyme and MgCl2 might be necessary to get consistent results.

4) 0.5% BSA is added to all solutions used after dejellying (i.e. 1x MMR for the post-dejellying rinse, the injection medium).Following dejellying, the unfertilized eggs are easily activated by mechanical stimuli—the BSA prevents the egg activation that often happens during the post-dejellying rinse and egg loading into the injection dishes.Omitting calcium from the dejellying and post-dejellying rinse solutions also helps to prevent the egg activation.

In addition, the BSA appears to maintain egg activity for fertilization prior to injection and aid in the healing of injected eggs.The underlying mechanisms are unclear, but the addition of BSA to the culture medium increases the number of transgenic embryos that gastrulate normally.

5) The plasmid backbones are fully removed from transgene constructs (Hirsh, N. et al., Dev Dyn. 225, 422-433, 2002).In the original Kroll and Amaya protocols, plasmids carrying a transgene construct are linearized and used directly in the transgenic reaction.However, we have found that the flanking vector sequence introduced with a transgene can interfere with correct expression of the transgene.This appears to be enhancer/promoter-specific, but in severe cases, strong background expression can mask the “correct” expression pattern of the transgene Thus, we completely remove the plasmid backbone sequences from a transgene cassette by restriction enzyme digestion followed by gel electrophoresis.The transgene is then purified from the agarose gel using the Qiaquick Gel Extraction kit (Qiagen), precipitated with ethanol once, and finally dissolved in 10 mM KCl when it is ready for use in transgenesis.

TOP: An embryo carrying a transgene in which the plasmid backbone has been left in the injected construct.

BOTTOM:An embryo carrying the same transgene but without the plasmid sequence.

STOCKS AND PREMADE BUFFERS

I. Stock Media

10 x MMR (with Ca2+) (1L)

from stock solutions

in grams

1 M NaCl

250 ml 4 M NaCl

58.44 g NaCl

20 mM KCl

20 ml 1 M KCl

1.49 g KCl

10 mM MgCl2

10 ml 1 M MgCl2

2.03 g MgCl2·6H2O

50 mM HEPES

50 ml 1 M HEPES

11.92 g HEPES

20 mM CaCl2

20 ml 1 M CaCl2

2.94 g CaCl2·2H2O

add water to 1 Liter, pH to 7.4-7.6 with NaOH

Autoclave if made from dry stocks. Otherwise just combine sterile solutions + sterile H2O in a sterile bottle.

10x MMR (No Ca2+)

Prepare as above, but omit 20 mM CaCl2.This reagent is used in steps prior to injection to prevent egg activation.

Dissolve 1.54g in 10 ml of sterile water. Store in aliquots of 66 µl at -20°C.

·10% bovine serum albumin (BSA)(Sigma A-3294)

Slowly dissolve 40 g by floating it on 375 ml of deionized water. It should slowly go into solution. Bring final volume to 400 ml and pH to 7.6 with KOH. Final solution should look yellowish. Store at -20°C in 4 ml aliquots.

For the "anesthetizing bath" make up at a concentration of 1:500 in distilled water plus 1g/L Instant Ocean salts and 1g/L Sodium Bicarbonate. You can also make it up in 1/10x MBS.Adjust pH to 7.4-7.6 and store in 4°C.

Preparation of sperm prep working solutions (to be made fresh each time & kept on ice)

2 X NPB(30 ml)

Stock

Final Concentration in 1X

10 ml

1.5M

Sucrose (-20ºC stock)

250 mM

900 µl

1M

HEPES (pH 7.7, thawed -20°C stock)

15 mM

120 µl

0.5 M

EDTA, pH 8.0 (RT stock)

1 mM

300 µl

100 mM

Spermidine (-20°C stock)

0.5 mM

120 µl

100 mM

Spermine (-20°C stock)

0.2 mM

60 µl

1 M

DTT (-20°C stock)

1 mM

18.5 ml

Sterile water

Adjust pH to 7.7 if necessary (use KOH).

1 X NPB (40 ml)

20 ml2X NPB

20 mlsterile H20

1 X NPB + 3% BSA (12 ml)

6 ml2 X NPB

2.4 mlsterile H20

3.6 ml10% BSA

1 X NPB + 0.3%BSA (6 ml)

3 ml2 X NPB

2.8 mlsterile H2O

180 µl10% BSA

Sperm Nuclei Storage Buffer (1 ml)

500 µl2 X NPB

300 µlglycerol

170 µlsterile H20

30 µl10% BSA

1 x MMR (50 ml)

5.0 ml10 x MMR (with Ca)

45 mldistilled water

Sperm nuclei preparation protocol

1) Anesthetize a male in 1:500 MS222 at room temperature (30-45 min).Make the above working solutions from the thawed components and place on ice.

2) After the frog is immobilized (about 30-45 minutes), remove two testes and place them into a 60 x 15 mm petri dish filled with ice-cold 1X MMR.

3) Clean testes (remove as much fat bodies and blood as possible - carefully poke holes in blood vessels and push blood out) under dissecting scope and rinse two times in ice-cold 1X MMR and two times in ice-cold 1X NPB.Clean as much as possible without damaging the testes and releasing sperm.

4) Put the cleaned testis in another dry petri dish and under a dissecting scope macerate the tissue well (until clumps are no longer visible to the naked eye) with two pairs of clean forceps.This can be done by gently grasping the testis with one pair of forceps and slowly teasing the other end with another pair of forceps.The entire macerating process should take around 15 minutes.

5) Resuspend the macerated testes in 2 ml of 1X NPB by gently pipetting the solution up and down through a fire-polished, truncated pasteur pipette or truncated plastic transfer pipette with an opening of about 3 mm in diameter.Next, gently pipette the solution up and down through a cut yellow pipette tip.Finally, use a regular yellow pipette tip to completely eliminate any aggregates.

6) Cut the snap cap from a 12 x 75 round bottom polypropylene tube (Falcon 2063) along its equator to make a ring of pastic.

Place the 2 x 2 cm square of Nitex mesh (100-110 µm, Nitex HC-3-110 etc.) across the opening of a 14 ml (17 x 100 mm) round bottom polypropylene tube (Falcon 2059).Secure the mesh by placing the plastic ring onto the mesh square and forcing it into the inner diameter of the tube.

Pass the sperm suspension through the mesh and into the tube using a pipettor with a cut blue pipette tip (we use round bottom polypropylene tubes; Falcon 2059).Rinse the forceps and dish with 8 ml of 1X NPB and force this through the mesh also.The sperm suspension may not flow through the homemade filter apparatus because of surface tension.Gently touch the mesh surface with the cut blue pipette tip to initiate the filtering.

7) Pellet the sperm by centrifugation at 3,000 rpm for 10 min. at 4°C (Sorvall HB-6 rotor).Don't be concerned if the pellet looks slightly reddish.Carefully aspirate off the supernatant without allowing the sperm pellet to come in contact with the air.Resuspend the sperm in 0.5 ml of 1 x NPB first by pipetting with the cut-wide and cut-narrow yellow tips.Then dilute to 8 ml NPB and repellet by centrifugation at 3,000 rpm, 10 min., 4°C.

8) Resuspend the pellet in 1 ml NPB with a cut plastic blue pipette tip, again not allowing the pellet to be exposed to air.Warm the suspension to room temperature.

13) Resuspend the pellet in 500µl sperm nuclei storage buffer using cut-off tips and transfer suspension into a 1.5 ml eppendorf tube.Count the sperm nuclei density using a hemacytometer (Fisher, cat. #: 02-671-5):dilute a small amount of the concentrated nuclei 1:100 in sperm dilution buffer and add 1µl of 1:100 Hoechst stock to visualize the sperm heads under a fluorescence microscope.For a 1:100 dilution of our sperm nuclei stock, we typically obtain counts of 75-125 (X104 cells/ml).At this concentration, the undiluted stock contains 75-125 sperm nuclei/nl.If your sperm nuclei stock is substantially less concentrated (i.e. a count of <50 for a 1:100 dilution), repellet the nuclei and resuspend in a smaller volume of sperm nuclei storage buffer.Sperm nuclei can be stored at 4°C and used for transplantations for up to 48 hours. The remained sperm nuclei can be stored at –80°C. Freshly prepared sperm nuclei are better, but the frozen nuclei also give satisfactory results. You can simply “slow freeze” the nuclei; ie-simply put them in the –80°C freezer. No need for liquid nitrogen, etc.

1) Sacrifice 2 or 3 females and remove the entire ovaries.If oocytes are mostly large and there are a lot, use only 2 females.If one or both females have a lot of tiny oocytes or not many, use an extra female.It is very important to have enough starting material for this prep!

2) Cut ovaries into small pieces using scissors and digest for approximately 1.5-3.5 hours at 28°C (in water bath with agitation) in 3 mg/ml collagenase made up in OR2.It takes less time to complete the digestion if you split the oocytes into a few containers with approximately 100-150 ml of collagenase+OR2 solution in each container.

3) Begin checking the oocytes every 15 minutes after about an hour-they'll be ready once you no longer see any clumps of oocytes.

4) After digestion, wash the oocytes several times in OR2-if you swirl them in a flask, the small immature oocytes will float to the top and you can easily pour some of them off.

5) Transfer oocytes to OR3. The oocytes will keep in OR3 for about a week at 16-18°C. Leave overnight and sort the next morning. You can also begin the sorting process by removing any dead, dying, or tiny oocytes to save time for the next day.

II. Extract prep

1) Remove as many dead, dying, or tiny oocytes as possible or use a transfer pipette with a cut-off tip to transfer good oocytes into a separate dish.This is the step that requires a lot of time and a lot of work.You have to work quickly, and it is not necessary to remove every single undesired oocyte.You should probably try to be completely finished with your sorting by noon at the latest.

2) Rinse oocytes four times in 1 x XB (approx. 100 ml each rinse)

3) Rinse oocytes two times in CSF-XB with protease inhibitors added just before use. (approx. 50 ml each rinse).

4) Load oocytes gently into 30 ml centrifuge tubes (open-top corex tube) using a cutoff transfer pipet. Remove as much CSF-XB as possible and try to balance tubes visually, with approximately equal volumes in each centrifuge tube. (You should have at least enough oocytes to half fill two 30 ml centrifuge tubes. It is probably better if you start with enough to fill each tube 3/4, depending on the quality of the oocytes.)

5) Add 1 ml Versilube F-50 on top of oocytes in each centrifuge tube.Versilube F-50 is trademarked by General Electric, and is same with MIL-S-81087C (F-50) which can be purchased from Andpak-EMA.

6) Spin tubes in a clinical centrifuge at room temperature for about 60 seconds at 1000 rpm (150g), then for an additional 30 seconds at 2000 rpm (600g). This will pack the oocytes, but they should not be lysed or broken.

7) Remove the excess CSF-XB and Versilube, which should cover the oocytes after the packing spin.

8) Spin at 4º C for 10 minutes at 10,000 rpm in the Sorvall RC-5B, using the HB-6 rotor (16342g). While this is spinning, set the ultracentrifuge to cool. (Set to 70,000 rpm, 4°C, 1.5 hours).

9) The previous spin should separate the oocytes into 3 layers: lipid(top), cytoplasm(center), and yolk and pigment(bottom). Any excess Versilube from previous steps will appear as bubbles in the cytoplasmic layer. (The cytoplasmic layer should make up more than half of the volume in the centrifuge tubes.)

10) Remove the cytoplasmic layer (which should be grey to brown/gold-but may be darker or lighter depending on the pigment of the oocytes; in general this does tend to be darker in the oocyte prep than in the egg extract) with a drawn out disposable glass pipet. (pull under a flame and cut off the tip).A 3-5 mL syringe with a long, bent needle also works very well. Transfer to a 15 ml graduated tube on ice to measure volume. (You should be able to fill your 15 ml centrifuge tube at least 3/4.)

12) Spin at 4ºC for 10 minutes at 10,000 rpm using the HB-6 rotor. Collect the cytoplasmic layer as described in step 10 and transfer to ultracentrifuge tubes (Beckman 349622).Fill each at least halfway, but not high enough to lose volume during a fixed angle spin.Balance tubes carefully.

13) Spin in ultracentrifuge at 4ºC for 1.5 hours at 70K rpm, no brake (set deceleration to zero) in the Beckman TLA100.4 rotor.After this spin you will have 4 layers, top to bottom: lipid, cytosol, membranes/mitochondria and glycogen/ribosomes.

1) Fill a Hamilton gas-tight syringe (25µl) and a length of tygon tubing (long enough to reach from the pump to the micromanipulator) with mineral oil. Use the plastic rod that comes with the micromanipulator as your "needle holder". The injection needle filled with your transgenic reaction is directly inserted into the tygon tubing, and the needle is fixed to the manipulator rod by tightening the screw on the rod.

2) Set the pump to the desired speed (18.0µl/hr, diameter: 0.73 mm for 25µl Hamilton Gas-tight Syringe). Wait for the backpressure to subside, start the pump, and inject at a rate of approximately one egg per second.Remember to check periodically for bubbles in the tubing and clogs in the needle.The needle can be intermittently lifted out of the injection media to remove any debris that gathers at the tip.

Preparation of injection needles

Preparation of proper needles is essential for the success of the transgenic procedure.The nuclear transplantation needles should have a long sloping taper from the widest part of the needle to the tip (12-15 mm) to control the flow rate through the large needle tip.The tip must be large enough to allow sperm nuclei to pass through unharmed, but small enough to prevent large wounds in the egg (70-80µm).The tip should be beveled to allow piercing of the cell membrane with as small an amount of damage as possible.

NOTE:Previous protocols have suggested using a siliconizing agent to coat injection needles to prevent shearing of sperm nuclei.Our experimental evidence suggests that this process is not necessary.

To produce the correct tip size and shape, place a pulled capillary over a stage micrometer under a dissection microscope, and clip the tip to 70-80µm.Two suggested methods to produce the appropriate bevel shape in the needle tip are:A) a pair of forceps that have uneven ends can be placed parallel to the axis of the needle and the end of the needle broken to the correct size, or B) two breaks can be made using a normal pair of forceps—the first to set the diameter of the needle tip opening, the second to tap off one “side” of the tip to produce the bevel.

Preparation of agarose coated injection dishes

Nuclear transplantations are performed in a petri dish that has a square depression within an agarose bed.To prepare dishes, pour molten agarose (1% agarose in 0.4x MMR with Ca2+) into several 60-mm Petri dishes.Before the agarose solidifies, float a square plastic plate (2.5 x 2.5 cm cutouts of plastic pipet tip boxes work well) on the surface so that a depression will be formed after the agarose solidifies.Ensure that no bubbles are present under the plastic plate—remove and refloat if necessary.After the agarose solidifies, carefully remove the plastic plates, wrap the openings in Parafilm, and store at 4°C.A typical dish will hold 300-400 X. laevis eggs.

Preparation of special tips

·Cut-off p200 tips to handle sperm nuclei

The tip should be cut with a clean razor blade to approximately 1 mm inside diameter.

Things to be prepared the day before the REMI transgenesis

pH to 7.5 with NaOH.Phenol Red may be added as a pH indicator (0.5 ml of 0.5 % solution (GIBCO-BRL, 15100-043)).Sterilize by filtration or use sterile water for the preparation.Store at 14°C.

Post-dejellying rinse solution (300 ml)

10 x MMR (no Ca2+)30 ml (final 1.0x)

10% BSA15 ml

distilled water255 ml

Sterilize by filtration or use sterile water.Store at 14°C.

Prepare female frogs

Inject HCG (400µl of 2 units/µl solution) to a female the night before the sperm nuclear transplantation (use 1 ml syringe with 27G needle).Keep the injected female in a 16°C incubator for overnight.Usually the frog start laying eggs 11 hours after the injection.

REMI transgenesis protocol

Throughout the following steps, use cut-off tips (see above) for all mixing steps involving sperm nuclei.Avoid making bubbles, which damage the sperm nuclei.Keep condensed sperm nuclei and oocyte extract on ice.SDB, MOH, and decondensed nuclei reactions should be at room temperature.Do not use centrifugation to collect sperm nuclei reactions to the bottom of tubes (it leads packing of the nuclei).

1) Thaw aliquots of SDB and MOH and equilibrate to room temperature.

2) Prepare fresh dejelly solution: 2.5% cysteine in 1x MMR (no Ca2+).Use the freebase form of cysteine and adjust the pH of the solution to 8.5 with NaOH.The same batch of dejelly solution can be used throughout the day.

3) If using a restriction enzyme in the transgenic reaction (see “Comments” section), dilute the appropriate enzyme 1:50 in SDB and place it on ice.

4) Thaw aliquots of oocyte extract and sperm nuclei on ice.

5) Squeeze eggs from the frog into High-Salt MBS.Around 1000 eggs are sufficient for one round of injection.

6) Prepare the REMI transgenic reaction. Gently mix 4-5µl of sperm nuclei (100 nuclei/nl) and 1µl DNA (100-150 ng) using cut-off p200 tips.If the sperm nuclei are fresh, use 4µl; if the nuclei are frozen, use 5µl.Incubate the reaction for 5 minutes at room temperature.For co-transgenesis, a 4:1 molar ratio of PCR product to GFP cassette is recommended.

7) To the reaction tube, add the following;

2µl oocyte extract

X µlSDB (varies depending on sperm nuclei and DNA concentration)

to a total volume of 18.5µl

If you are including restriction enzyme in the reaction, also add 1µl of 100 mM MgCl2 and 0.5µl of diluted restriction enzyme to the reaction mixture but keep the final volume 18.5µl.

Mix gently using a cut-off p200 tip and incubate for 15 minutes at room temperature. During the incubation step, proceed to steps 8,9, and 10.

8) Dejelly eggs. Remove as much High-Salt MBS as possible from the dish. Pour the dejellying solution onto the eggs and gently swirl a few times.After a few minutes (2-4 minutes), the eggs will come free from the dish bottom and pack tightly.Do not swirl vigourosly, as too much mechanical stress will prematurely activate the eggs.

9) Gently wash dejellied eggs 3-4 times with 30-50 ml of the post-dejellying rinse solution (1x MMR (no Ca2+) + 0.5% BSA).Transfer them using the cutoff tip of a plastic transfer pipette—be gentle during this process and do not expose the eggs to air or surface tension.

10) Carefully load the eggs into injection dishes filled with the injection medium.It is important to not overfill the dishes, as mechanical stress from packing can lead to gastrulation defects—ideally all eggs should maintain a round shape.If they are misshapen in any way when the dish is lying flat, they are packed too tightly!To avoid overpacking, place an injection dish at an angle by setting it on its top, and use a transfer pipet to fill the dish ~2/3 full of eggs.Carefully tilt the dish in the opposite direction and gently tap to disperse the eggs evenly over the well.

11) Add 4.5µl of the transgenic reaction to 166µl of MOH.DO NOT MIX IMMEDIATELY.Allow the mixture to equilibrate for 2-3 minutes while you are preparing the injection setup.This dilution gives a final concentration of 3 nuclei/5 nl.

Before loading, mix gently 5-10 times using a cutoff p200 tip.Do NOT introduce air bubbles. The decondensed sperm nuclei are very fragile!

12) Load the diluted REMI reaction into injection needles by drawing up ~75µl of the transgenic reaction into a “loading tip”.Attach an empty needle to the Tygon tubing on the loading tip and gently backfill the needle.Remove the entire loading tip from the pipetteman (with the needle attached), and then remove the needle from the end of the loading tip.Insert the reaction-filled needle into the mineral oil-filled Tygon tubing connected to the Hamilton syringe pump ensuring that there are no air bubbles introduced into the line.Fix the needle to the rod of your micromanipulator.

13) Place an egg-filled injection dish under the microscope, and ensure that the pump settings are correct (speed: 18.0µl/hr, diameter: 0.73 mm for 25µl Hamilton Gas-tight Syringe).Position the needle tip under the surface of the medium, and turn on the pump.Focus on the needle tip to ensure that there is proper flow—if not, slowly depress the plunger of the pump to force any air bubbles out of the needle tip.If there is still no flow, load a new needle.

14) Inject eggs at a rate of approximately one egg per second.This should result in the injection of 3 nuclei / egg—this has empirically yielded optimal results. The transgenic reaction gradually loses activity, so ensure that all injections are completed within 45 minutes!

15) Place the eggs in a 16°C incubator for 3 hours until they reach 4-cell stage.This temperature has been determined to maximize the number of healthy embryos.

16) The 4 to 8-cell stages are the best for sorting eggs.Transfer only embryos with a normal cleavage pattern to 6-well dishes filled with culture medium (0.3x MMR (with Ca2+) + 0.5% BSA (with gentamycin).Put 15-20 embryos in each well of the dish.Note that it is not necessary to coat these dishes with agarose as the medium contains BSA.Approximately 30% of injected eggs will cleave normally at this point.

17) Incubate at 16-18°C overnight (17°C is the best).At temperatures higher than 18°C, injected embryos are much more susceptible to gastrulation defects and have reduced survival rates.

18) Next morning, remove dead and abnormal embryos before they start gastrulation.

19) It is important to not disturb the embryos when they are gastrulating.The embryos are particularly sensitive to changes in temperature during gastrulation.You may remove dead or exogastrula embryos after the gastrulation if necessary.

20) Following gastrulation, the BSA-containing medium is not necessary and the embryos can be cultured as normal until they reach desired stages.